RNA structure interactions and ribonucleoprotein processes of the influenza A virus

Abstract

In one more years, we will 'celebrate' an exact centenary of the Spanish flu pandemic. With the rapid evolution of the influenza virus, the possibility of novel pandemic remains ever a concern. This review covers our current knowledge of the influenza A virus: on the role of RNA in translation, replication, what is known of the expressed proteins and the protein products generated from alternative splicing, and on the role of base pairing in RNA structure. We highlight the main events associated with viral entry into the cell, the transcription and replication process, an export of the viral genetic material from the nucleus and the final release of the virus. We discuss the observed potential roles of RNA secondary structure (the RNA base-pairing arrangement) and RNA/RNA interactions in this scheme.

Figure 1.

A cartoon representation of the transcription process. The figure shows the time-dependent and structural relationships of the vRNP and associated viral polymerase, which consists of the subunits PB1, PB2 and PA (from genome Segments 2, 1 and 3, respectively). The viral polymerase binds to the partially complementary 5′ and 3′ termini of the vRNA segment. The progression is from top to bottom. (Top) The vRNP has entered the nucleus of the host cell and has separated from other vRNPs. (Next step) The transcription is initiated by PB2 hijacking a 5′ cap from the host pre-mRNA, followed by endonucleolytic cleavage of the host pre-mRNA by PA. (Next frame) Transcription begins from the 3′ end of the vRNA sequence with the spliced-in primer sequence and cap merged onto the forming mRNA sequence. (Next frame) Transcription continues with the viral polymerase somehow dislodging the NP in its wake to obtain the mRNA transcript. Also depicted faintly are alternative splice factors from the spliceosome that can influence the resulting transcription. (Next frame) While the transcription process is happening, the vRNA and NP tend to recombine back to the rest state. (Final step) The poly-adenylation step is reached where further editing is required of the structure using the host RNA polymerase II system. It is important to remember that during this entire process, various mRNA packaging proteins are being recruited (e.g. the CBP. These are shown as a faint covering of the final mRNA. All this packaging is required for transport to the cytosol for translation into viral proteins. The schematic is inspired from [44] and [42]. The structure of the vRNP is based on the right-handed helix in [42]; however, it is important to remember that [43] reports a left-handed helix. (A colour version of this figure is available online at: https://academic.oup.com/bfg)

Figure 2.

A cartoon representation of the replication process showing the time-dependent relationship between the vRNP and trans-acting viral polymerase (PB1, PB2 and PA). Note that this model is still under investigation and the possibility remains that replication is also done in cis. The progression of the figure is from top to bottom. (Top) The vRNP has entered the nucleus of the host cell and has separated from other vRNPs. Perhaps at this stage, the vRNP has become anchored to chromatin, though this is not specified. (Next step) The 3′ terminus of the vRNA segment somehow unwinds and the trans-acting viral polymerase captures this free segment and begins copying the sequence in the 3′–5′ direction, making an antisense copy of whatever template is present. (Next frame) The process continues with the viral polymerase somehow unwinding the vRNA + NPs, all the while recruiting new NPs for the copy. (Next frame) When the replication is complete, the structure is eventually released. There are several steps in the trans-acting model that are not fully determined yet. The schematic is inspired from [44] and [42]. The structure of the vRNP is based on the right-handed helix in [42]; however, it is important to remember that [43] reports a left-handed helix. (A colour version of this figure is available online at: https://academic.oup.com/bfg)

Figure 3.

A scan of the thermodynamic stability of the cRNA secondary structure (variability of the structures) for the influenza A viral genome (M, Segment 7) where the largely universally conserved segments reported in [87] are indicated by the long semitransparent gray/green boxes (also marked by dark arrows, bottom). The graph spectrum was generated using Genepoem [90] and calculated using vs_subopt/vswindow [89] using default settings (window size 200 nt, Kuhn length 4 nt). The very dark gray/blue boxes indicate regions of highly stable RNA secondary structure (not only hairpins but even a region of stability). The very short gray/magenta boxes indicate regions where a stable loop is present. The particular strains that were calculated are shown on the right side of each free energy spectrum. The sequences were obtained from the influenza database at https://www.fludb.org [88]. (A colour version of this figure is available online at: https://academic.oup.com/bfg)

Figure 4.

The same calculation as Figure 3 of the thermodynamic stability of the vRNA (negative strand) secondary structures. The details of the figure are explained in the caption of Figure 3. The settings and sequences used in the calculation are the same as in Figure 3. (A colour version of this figure is available online at: https://academic.oup.com/bfg)

Figure 5.

Similar to Figures 3 and 4, a calculation of thermodynamic stability of the vRNA (negative strand) secondary structure for the kissing loop regions for H5N2 in Segments 2 and 8, as reported in [83]. The cRNA sequence was obtained from https://www.fludb.org and converted to vRNA. The details of the spectrum are explained in the caption of Figure 3, and the settings used in the calculations are the same as Figures 3 and 4. The very dark gray/blue boxes indicate regions of highly stable RNA secondary structure and the very short gray/magenta boxes indicate regions where stable hairpin loops are present. The connected-semitransparent gray/green boxes indicate the band where the kissing loop hairpins are found in Segments 2 and 8 of the strain, and a dotted line is shown to indicate the respective interaction between these two vRNP segments. (A colour version of this figure is available online at: https://academic.oup.com/bfg)